The poly(ADP-Ribose) polymerase inhibitor ABT-888 reduces radiation-induced nuclear EGFR and augments head and neck tumor response to radiotherapy - PubMed (original) (raw)

The poly(ADP-Ribose) polymerase inhibitor ABT-888 reduces radiation-induced nuclear EGFR and augments head and neck tumor response to radiotherapy

Somaira Nowsheen et al. Radiother Oncol. 2011 Jun.

Abstract

Background and purpose: Current therapies for head and neck cancer frequently are not curative, necessitating novel therapeutic strategies. Thus, we studied whether inhibition of poly(ADP-Ribose) polymerase (PARP), a key DNA repair enzyme, could improve efficacy of radiotherapy in human head and neck cancer.

Materials and methods: UM-SCC1, UM-SCC5, UM-SCC6, and FaDu human head and neck cancer cellular susceptibility to the PARP inhibitor (PARPi) ABT-888 and/or radiation (IR) was assessed using colony formation assays. DNA damage was evaluated using the alkaline comet assay and immunostaining for γ-H2AX foci. Non-homologous end-joining (NHEJ) mediated repair was measured using phospho-DNA-Pk foci. Epidermal growth factor receptor (EGFR) location was assessed by immunostaining. Poly ADP-Ribose polymerization (PAR) levels were assessed using immunoblotting.

Results: Human head and neck cancer cells exhibited enhanced cytotoxicity with IR and ABT-888 compared to either agent alone. This increased susceptibility correlated with reduced nuclear EGFR, attenuation of NHEJ, and persistence of DNA damage following IR. Interestingly, a subset of head and neck cancer cells which had elevated basal PAR levels was susceptible to PARPi alone.

Conclusions: Combining radiotherapy and PARP inhibition may improve outcomes and quality of life for head and neck cancer patients treated with radiotherapy. Furthermore, this novel strategy may also be feasible in other tumor types. Moreover, PAR levels should be investigated as a potential biomarker for tumor susceptibility to PARP inhibition.

Published by Elsevier Ireland Ltd.

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Conflict of interest statement

Conflict of interest statement

The authors declare no conflict of interest.

Figures

FIGURE 1

FIGURE 1. IR augments head and neck tumor susceptibility to the PARP inhibitor ABT-888

Combination of IR and ABT-888 reduces the viability of (A) UM-SCC1, (B) UM-SCC5, (C) UM-SCC6, and (D) FaDu head and neck cancer cells. Shown is the representative data of at least 3 independent experiments of the cell viability following various treatments as measured by colony formation assay, corrected for the survival fraction following RT (mean +/− SEM, *p<0.01, **p<0.001). Interestingly, UM-SCC1 and UM-SCC5 demonstrate susceptibility to ABT-888 alone.

FIGURE 2

FIGURE 2. Persistent DNA damage is observed in head and neck cancer cells treated with ABT-888 and radiation

The inset in (A) is a representative image of UM-SCC1 cells exhibiting comet tail following IR. IR enhances the mean tail moment, indicative of total DNA damage (SSB and DSB) in ABT-888 treated (B) UM-SCC1 and (C) UM-SCC6 cell lines, as measured by the alkaline comet assay. Addition of ABT-888 inhibits resolution of IR-induced DNA damage. The inset in (D) is a representative image of UM-SCC1 cells exhibiting γ-H2AX foci, a commonly used marker for DSBs, following IR. The comet data correlates with the number of cells with DSBs in (E) UM-SCC1 and (F) UM-SCC6 cells as evidenced by persistent γ-H2AX foci. Shown is the representative data of 3 independent experiments the % of cells (mean +/− SEM) with >10 foci (*p<0.05, **p<0.01).

FIGURE 3

FIGURE 3. ABT-888 attenuates IR-induced non-homologous end joining (NHEJ) in head and neck cancer cells

The inset in (A) is a representative image of UM-SCC1 cells exhibiting DNA Pk T2609 foci, well characterized markers of NHEJ-mediated DNA DSB repair, following IR. ABT-888 attenuates IR-induced phosphorylated DNA Pk foci in (B) UM-SCC1, (C) UM-SCC5, (D) UM-SCC6, and (E) FaDu cells. Shown is the representative data of 3 independent experiments the % of cells (mean +/− SEM) with >10 foci (*p<0.05, **p<0.01).

FIGURE 4

FIGURE 4. ABT-888 reduces nuclear epidermal growth factor receptor (EGFR) following radiation

The inset in (A) is a representative image of UM-SCC6 cells exhibiting nuclear (N), both nuclear and cytosolic (NC), and cytosolic (C) EGFR. EGFR location was analyzed following IR and cells were assessed as having predominantly nuclear staining, predominantly cytoplasmic staining, or mixed nuclear/cytoplasmic staining. ABT-888 blocks nuclear translocation of EGFR following IR in (B) UM-SCC1, (C) UM-SCC5, (D) UM-SCC6, and (E) FaDu cells. Shown is the representative data of three independent experiments the % of cells (mean ± SEM) with strictly nuclear (N), strictly cytosolic (C), or mixed nuclear/cytosolic (NC) EGFR staining (*p < 0.05, **p < 0.01).

FIGURE 5

FIGURE 5. Basal PAR level correlates with susceptibility of head and neck cancer cells to ABT-888

Basal PAR levels in head and neck cells were analyzed following ABT-888 treatment and correlated with susceptibility to ABT-888. Shown is a representative western blot of at least 3 independent experiments.

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